The objective of this Phase II Research Proposal is to continue the successfully completed work of the Phase I Research project by further developing affinity-based mass spectrometry assays for the detection/characterization of Biodefense agents, specifically the family of Staphylococcal Enterotoxins (SE). The assays will utilize affinity capture in pipette-fitted microcolumns modified with anti-SE antibodies to selectively retrieve SE from buffer, tap water, and milk samples. Detection will be achieved by measuring the molecular weight of the eluted SE via MALDI-TOF mass spectrometry. The assay platform is unique as it offers dual specificity: the first comes from the use of a capturing antibody, and the second comes from the observation of a specific m/z value, characteristic of the analyzed toxin, in the resulting mass spectra. The use of polyclonal antibodies in combination with MS enables capture and detection of protein toxins and related variants in a single assay, and in the process significantly reduces the cost and time of analysis. In addition, multiple capture antibodies can be used on a single device, providing a multiplexed assay array able to screen for multiple biodefense agents in a single analysis, with each species differentiated in the mass spectrum by their unique m/z. In such, the proposed assay offers significant advantages over existing and commercially available sandwich-based assays. [unreadable] [unreadable] Under the four specific aims of this proposal, assays for the detection of the SE will be developed and their reproducibility and sensitivities demonstrated. Quantitative approaches for the detection of the SE via the proposed affinity-based mass spectrometric assay will be developed and applied. Upon successfully developing individual assays for the SE, a comprehensive multiplexed assay will be developed to provide the means to simultaneously detect multiple enterotoxins in a single analysis. In addition, routines will be developed, of which when used in combination with data evaluation software, will guide workflow in depicting samples containing SE. Additionally, secondary analyses that are able to assist in the characterization of variant forms of the enterotoxins will also be developed. The successful completion of these Phase II Specific Aims will result in fully-functional SE-MSIA assays/multiplexed assay capable of detecting/characterizing a family of toxins and a proteomics-based platform for the detection of SE to be offered to a network of laboratories (e.g., members of the CDC's Laboratory Response Network). [unreadable] [unreadable] We view the most direct social benefit, with respect to public health, to be the ability to detect Biodefense Agents present in water/milk/food supplies on a routine basis. In the event of no-detection, there is a corresponding "peace of mind" that accompanies the knowledge that such routine assays are in place - akin to periodic medical exams revealing an individual is healthy. In the event of detection, the social benefit stems from the ability to take corrective measures to retard the spread of contaminated sources, which clearly has the ability to save human lives. [unreadable] [unreadable] [unreadable]